The present invention generally provides peptides that comprise a recognition sequence motif for phosphotyrosine binding proteins. In particular, the present invention provides peptides which comprise a core sequence of amino acids, and analogs thereof, which are recognized and bound by the PTB phosphotyrosine binding domain. Also provided are methods of using the peptides of the invention in diagnostic, screening and therapeutic applications.
Receptor signaling pathways are the subject of widespread research efforts. A better understanding of these signaling pathways will lead to the design of new and more effective drugs in the treatment of many diseases. Of particular interest are the growth factor and related receptor signaling pathways and their role in cell growth and differentiation. Binding of a particular growth factor to its receptor on the cell plasma membrane can stimulate a wide variety of biochemical responses, including changes in ion fluxes, activation of various kinases, alteration of cell shape, transcription of various genes and modulation of enzymatic activities in cellular metabolism.
In particular, upon binding an external ligand, a receptor may undergo auto-phosphorylation of specific tyrosine residues, and/or may phosphorylate other proteins. This tyrosine phosphorylation creates binding sites for cytoplasmic signaling proteins which have specific domains that recognize the phosphorylated tyrosine and adjacent residues. Once bound, these signaling proteins may in turn be activated. The activated signaling proteins then may effect downstream processes. Pawson and Gish, Cell 71:359-362 (1992).
Src Homologous, or "SH2" domains are amino acid sequences that are similar to a 100-residue, non-catalytic region of the Src tyrosine kinase and are present in various signaling molecules. Sadowski et al., Mol. Cell. Biol. 6, 4396 (1986). SH2 domains are functional protein motifs that bind tyrosine-phosphorylated targets by recognizing phosphotyrosine and specific adjacent residues. J. A. Escobedo et al., Mol. Cell. Biol. 11, 1125 (1991); L. C. Cantley et al. Cell 64, 281 (1991); T. Pawson and G. D. Gish Cell 71, 359 (1992); S. Zhou et al. Cell 72, 767 (1993); G. Waksman, S. E. Shoelson, N. Pant, D. Cowburn, J. Kuriyan Cell 72, 779 (1993). Activation of tyrosine kinases by growth factors, cytokines, and oncogenic agents therefore serves as a switch for assembling SH2 domain-containing proteins with their tyrosine-phosphorylated targets in signaling complexes, in which downstream effectors are activated.
The use of phosphotyrosine binding domains, including SH2 domains, has been discussed in methods for identifying targets of tyrosine kinases in cells, and thus identifying intermediates in cell signaling pathways. See, PCT Patent Application No. WO 92/13001, to Schlessinger et al.
The specific use of SH2 domains and subdomains in affecting the SH2/phosphorylated ligand regulatory scheme, or screening for compounds which affect SH2 binding in this regulatory scheme, has been previously described. See, U.S. Pat. No. 5,352,660 to A. J. Pawson. The use of these domains in assaying for the presence of SH2 binding phosphoproteins has also been described.
Specific SH2 containing proteins include the products of the SHC gene. The SHC (which stands for SH2, Collagen) gene encodes a transforming protein, expressed as 46- and 52-kD proteins that are tyrosine phosphorylated in response to a number of growth factors, e.g., PDGF, EGF and FGF, and have been implicated as mediators of signaling from growth factor receptor and non-receptor tyrosine kinases to Ras. G. Pelicci et al. Cell 70, 93-104 (1992); M. Rozakis-Adcock et al. Nature, 360:689 (1992).
Thus, a great deal of attention has been directed toward studying these SH2 domains and their role in cell signaling pathways. However, SH2 domains, and the proteins which comprise them, are not the only phosphotyrosine binding mediators of such pathways.
A new phosphotyrosine binding ("PTB") domain has been identified within the sequence of the SHC protein. See, Kavanaugh and Williams, Science (1994) 266:1862-1865. This PTB domain was reported to specifically bind the tyrosine phosphorylated version of a target protein, which target protein was phosphorylated upon cell activation/stimulation, e.g., anti-IgM stimulated B cells, IL-6 stimulated HepG2 hepatoma cells, LIF stimulated CCE embryonic stem cells. The amino acid sequence of this domain is unlike that of any member of the known SH2 domain family. Therefore, although the nature of phosphotyrosine binding by the PTB domain is similar to that of the SH2 domain, functionally, and mechanistically, the two are very different.
The study of these cell signaling pathways, and the ability to control them requires identification and characterization of proteins which contain phosphotyrosine binding domains and the protein sequences to which they bind. The present invention meets these and other needs.